Axonal Degeneration Followed By Campylobacter Jejuni Lulei Hartley Guinea Pig Model

Objective: To establish the animal model of experimental peripheral nervedamage induced by intragastric administration of Campylobacter jejuni luleistain and to supply animal model for further study of the pathogenesis andimmunotherapy of human Gillan-Barre’s disease.Methods：1. Preparation of Bacterial suspension: Campylobacter jejuni was isolatedfrom the Guillain-Barre syndrome patients of North China and kept in theNeurology Laboratory of Second Hospital of Hebei Medical University. Takethem from the refrigerator in which the temperature is-80℃and add100ulof them to5ml of the broth with the condition of42℃micro-aerophilicatmosphere (10%CO2、5%O2、85%N2) for24hours. Passage the strains toColumbia blood plate which contains7%sterile and fiber blood and culturedunder the same conditions for24hours. Scrape the colonies after purification,and prepare to get the bacterial suspension.2. Identification of Campylobacter jejuni: the gram stain, peroxidase test,hippuric acid hydrolysis test.3. Experimental groups：48Hartley guinea pigs with the weight of4-6weeks of age,250-300g, SPF level of healthy and each half of male andfemale.36of them were experimental group and which were divided into fedwith live bacteria group (group A), fed with dead bacteria group (B group),fed with immune group (C group) with12in each.12was the control group(group D).4. Administration and concentration of the bacteria: The experimentalgroups were fed with2ml of bacterial suspension with3×108cfu/ml of theconcentration. The control group was orally fed with2ml of saline. Theanimals were banned of the diet but water for12hours before intragastric administration.5. Observation of the experimental animal: Since the date of feeding, theHartley guinea pig were checked daily of diet, stool, weight and physicalactivity.6. Stool culture: Isolate Campylobacter jejuni from the stools of allexperimental animals after feeding24hours.7. Dissection of the experimental animal: After weighing the animals,intraperitoneal anesthesia with4%chloral hydrate (10ul/g). Then disinfect theskin, opened the chest and perfuse the animals with4%paraformaldehyde.After perfusion remove bilateral sciatic nerve, brachial plexus nerve, brain,spinal cord, and fix all the tissues with4%paraformaldehyde for24hours.Then the tissues were taken for the neuropathological examination. In the endof the experiment, the experimental animals were managed by the NeurologyLaboratory of Hebei medical university.8. Neuropathological examination: the HE staining, immunohistochemistry,osmium tetroxide acid staining.Results:1. Clinical observation of the experimental animal: Each of group A3andgroup A4had symptoms of reduced activity, loss of appetite and weight loss,accounted for5.6%of the total (2/36). The rest of the experimental groups andthe control group animals did not appear obvious clinical symptoms.2. Peripheral neuropathy:Live bacteria feeding group (A group): All guinea pigs in the continuousfeeding for2weeks,3weeks,4weeks had pathological changes of peripheralnerve. The result of the HE staining of nerve was fiber structure, axonalswelling. Immunohistochemistry: mouse anti-human neurofilamentmonoclonal antibody of neurofilament showed different thickness andsegmental atrophy; rabbit anti-human myelin basic protein and myelin sheaththickness uniform and no demyelination. Osmic acid staining: the nervemyelin collapse and visible. One guinea pig of the continuous feeding5weeksgroup had the above pathological changes, HE staining of the rest two showed normal nerve fiber structure and no obvious pathological changes.Dead bacteria feeding group (B group): None of the experimental animalsshow the pathological staining change.Immune feeding group (C group): One guinea pig of the feeding for2weeks and3weeks in the experimental group have the above pathologicalchanges, all guinea pigs of the feeding for4weeks group appearedpathological changes, the rest had no obvious pathological changes.The control group (group D): All the experimental animals had nervepathological staining changed.3. Stool culture: All the stools of the experimental animals were negativein the stool culture before before the experimentation. Campylobacter jejuniwas isolated from all Group A experimental animals which was fed with livebacteria for24hours. The stool culture of other groups was negative.Conclusion:1. The gastrointestinal tract of the level of SPF healthy guinea pigs did nothave Campylobacter jejuni.2. Feeding Hartley guinea pig on Campylobacter jejuni can causeperipheral neuropathy. Fed with live bacteria group was better than fed withimmune group. Fed with simple dead bacteria did not cause guinea pigsperipheral neuropathy. Hartley guinea pig can be used as animal model for thestudy of Gillan-Barre’s disease.